Reference gene selection for qPCR analysis during somatic embryogenesis in longan tree
Reference gene selection for qPCR analysis during somatic embryogenesis in longan tree
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DOI：
10.1016/j.plantsci.2010.02.005
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发表时间：
2010-04-01
期刊：
影响因子：
5.2
通讯作者：
Lai, Z. X.
中科院分区：
文献类型：
--
作者：
Lin, Y. L.;Lai, Z. X.
实时逆转录PCR是研究植物体细胞胚胎发生（SE.）过程中基因表达相关变化的有力工具：但该方法缺乏理想的参考基因。选择最稳定的S.E.研究中，7个常用的参考基因（18S RNA，eIF-4a，UBQ ACTB，EF-1a，Histone H3和2-TUB）和功能基因（Fe-SOD. Cu/Zn-SOD和Mn-SOD）在20 ℃、25 ℃和30 ℃温度下对龙眼树胚性愈伤组织不同发育阶段的同步化培养进行了测定。这10个候选基因在龙眼衰老过程中的表达量变化不大。在25 ℃下最稳定的基因表达分析组合是UBQ和EF-1a或Fe-SOD和UBQ。然而，这些基因的表达在不同发育阶段和不同温度下有很大差异。利用geNorm、BestKeeper和Normandy软件包对结果进行综合分析，结果表明，UBQ和Fe-SOD可以作为龙眼S.E.各个时期基因表达分析的内对照。在不同温度下培养。(C)2010爱思唯尔爱尔兰有限公司版权所有。
Real-time reverse transcriptase PCR is a powerful tool to investigate relevant changes in gene expression during plant somatic embryogenesis (SE.): however, this method lacks ideal reference genes. To select the most stable reference genes for S.E. studies, the expression profiles of seven frequently used reference genes (18S RNA, eIF-4a, UBQ ACTB, EF-1a, Histone H3, and 2-TUB) and functional genes (Fe-SOD. Cu/Zn-SOD, and Mn-SOD) were tested in synchronized longan tree embryogenic cultures at different developmental stages at temperatures of 20 degrees C, 25 degrees C, and 30 degrees C. The expression of the 10 candidate genes showed little variation during longan S.E. at 25 degrees C. The most stable combination for gene expression analysis was UBQ and EF-1a or Fe-SOD and UBQ. However, the expression of these genes varied considerably at different developmental stages at different temperatures. Comprehensive analysis of the results using the software packages geNorm, BestKeeper, and NormFinder revealed that UBQ and Fe-SOD together could be used as internal controls for gene expression analysis in a wide variety of stages of longan S.E. cultured under different temperatures. (C) 2010 Elsevier Ireland Ltd. All rights reserved.